Cell and toxicant specific phosphorylation of conexin43: effects of lindane and TPA on rat myometrial and WB-F344 liver cell gap junctions

Previous studies showed that the pesticide lindane (gamma-hexachlorocyclohexane) inhibits gap junction intercellular communication in rat myometrial cells. The present study tested the hypothesis that lindane and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibit gap junction communication in rat myometrial and liver WBr-F344 cells by the common mechanism of increasing phosphorylation of the gap junction protein connexin43. We evaluated changes of connexin43 phosphorylation using Western blot of standard SDS-PAGE gels and cell immunostaining, and we monitored gap junction communication using microinjection and transfer of Lucifer yellow dye. Exposure of rat myometrial cells to lindane or TPA nearly abolished dye transfer but did not alter the electrophoretic mobility of connexin43, and neither lindane nor TPA increased phosphorylation of connexin43 as assessed by immunoblot with anti-phospho-connexin43 (S368) antibody. However, TPA increased punctate immunofluorescence staining of phospho-connexin43 (S368) in myometrial cells whereas lindane had no such effect. In WBr-F344 cells, lindane and TPA inhibited dye transfer. Lindane increased immunostaining for phospho-connexin43 (S368) in WBr-F344 cells without altering the abundance, electrophoretic mobility or phosphorylation of connexin43 as detected in immunoblots. TPA intensified a slower migrating connexin43 band and increased phospho-connexin43 (S368) in immunoblots, and intensified phospho-connexin43 immunostaining at WBr-F344 cell interfaces and nuclear regions. These results show that phosphorylation of connexin43 at serine 368 occurred in cell and toxicant specific manners and was independent of changes in electrophoretic mobility in standard SDS-PAGE gels. Moreover, lindane inhibited gap junction communication in myometrial cells by a mechanism that was not be explained by changes in phosphorylation of connexin43.     

Effect of thioridazine on gap junction intercellular communication in connexin 43-expressing cells

Propagation of electrical activity between myocytes in the heart requires gap junction channels, which contribute to coordinated conduction of the heartbeat. Some antipsychotic drugs, such as thioridazine and its active metabolite, mesoridazine, have known cardiac conduction side-effects, which have resulted in fatal or nearly fatal clinical consequences in patients. The physiological mechanisms responsible for these cardiac side-effects are unknown. We tested the effect of thioridazine and mesoridazine on gap junction-mediated intercellular communication between cells that express the major cardiac gap junction subtype connexin 43. Micromolar concentrations of thioridazine and mesoridazine inhibited gap junction-mediated intercellular communication between WB-F344 epithelial cells in a dose-dependent manner, as measured by fluorescent dye transfer. Kinetic analyses demonstrated that inhibition by 10 μmol/L thioridazine occurred within 5 min, achieved its maximal effect within 1 h, and was maintained for at least 24 h. Inhibition was reversible within 1 h upon removal of the drug. Western blot analysis of connexin 43 in a membrane-enriched fraction of WB-F344 cells treated with thioridazine revealed decreased amounts of unphosphorylated connexin 43, and appearance of a phosphorylated connexin 43 band that co-migrated with a “hyperphosphorylated” connexin 43 band present in TPA-inhibited cells. When tested for its effects on cardiomyocytes isolated from neonatal rats, thioridazine decreased fluorescent dye transfer between colonies of beating myocytes. Microinjection of individual cells with fluorescent dye also showed inhibition of dye transfer in thioridazine-treated cells compared to vehicle-treated cells. In addition, thioridazine, like TPA, inhibited rhythmic beating of myocytes within 15 min of application. In light of the fact that the thioridazine and mesoridazine concentrations used in these experiments are in the range of those used clinically in patients, our results suggest that inhibition of gap junction intercellular communication may be one factor contributing to the cardiac side-effects observed in some patients taking these medications.     

Functional Effects of Casein Kinase I-Catalyzed Phosphorylation on Lens Cell-to-Cell Coupling

The functional consequence of the casein kinase I-catalyzed phosphorylation of the lens gap junctional protein connexin49 was investigated using a sheep primary lens cell culture system. To determine whether the phosphorylation of connexin49 catalyzed by endogenous casein kinase I results in an altered junctional communication between lens cells, the effect of the casein kinase I-specific inhibitor CKI-7 on Lucifer Yellow dye transfer between cells in the lens culture was examined. Dye transfer was analyzed in cultures of different ages because we have demonstrated previously that the expression of connexin49 increases as the cultures age while that of connexin43, which is likely not a substrate for casein kinase I, has been shown to decrease [Yang & Louis (1999) Invest. Ophthalmol. Vis. Sci. 41: 2568–2564]. In 9-day old lens cultures, in which gap junctions are composed primarily of connexin43, CKI-7 had little effect on the rate of dye transfer between lens cells. In contrast, treatment of 15-day and 28-day old cultures with CKI-7 resulted in a significant increase in the rate of dye transfer. Thus, the extent of this CKI-7-dependent increase in cell-to-cell communication was positively correlated with the level of expression of connexin49, the major casein kinase I substrate in lens plasma membranes. These results suggest that the casein kinase I-catalyzed phosphorylation of connexin49 decreases cell communication between connexin49-containing gap junctions in the lens. Received: 31 July 2000/Revised: 12 January 2001     

Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B?

12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the protein-phosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidyl-prolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells.     

A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.

Gap junction-mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast growth factor (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of embryonic chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions. FGF induced sustained activation of extracellular signal-regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.     

Ilimaquinone inhibits gap junctional communication in a connexin isotype-specific manner

Ilimaquinone (IQ) and brefeldin A (BFA) disrupt the Golgi complex structure and block protein transport to the plasma membrane, and inhibit gap junctional communication. HeLa cells expressing rat connexin26, 32, or 43, or mouse connexin31, 36, 45, or 57, were used to study the response patterns of gap junctional communication (dye transfer) to ilimaquinone, brefeldin, and the potent protein kinase C (PKC) activator 12-O-tetradecanoylphorbol-13-acetate (TPA). 12-O-Tetradecanoylphorbol-13-acetate (followed for 2 h) caused dose- and time-dependent decreases in communication for five of seven connexins, the unresponsive being connexin45 and 57. Brefeldin (followed for 6 h) caused dose- and time-dependent decreases in communication for six of seven connexins, the exception being connexin26. These results are consistent with Golgi-mediated transport to the cell membrane for all connexins except connexin26. In contrast, ilimaquinone (followed for 6 h) caused a rapid (15-30 min) and nearly complete inhibition of dye transfer through connexin43 channels. For the other connexins, there was a slow and weak response for connexin26, 31, and 32, reaching 65-70% of control communication level, while connexin36, 45, and 57 were unresponsive. Thus, among the tested connexins, ilimaquinone has a strong specificity for connexin43, and the mechanism appears independent of the Golgi complex and of protein kinase C.     

SRC utilizes Cas to block gap junctional communication mediated by connexin43

The Src tyrosine kinase phosphorylates Cas (Crk-associated substrate) to confer anchorage independence and invasive growth potential to transformed cells. Gap junctional communication is often lower between aggressive tumor cells compared with normal or benign precursors. The gap junction protein connexin43 (Cx43) is a tumor suppressor that can inhibit tumor cell growth. Src can phosphorylate Cx43 to block gap junctional communication between transformed cells. However, mechanisms by which this event actually closes intercellular channels have not been clearly defined. Here, we report that Src and Cas associate with each other at intercellular junctions. In addition, Cas is required for Src to reduce dye transfer and electrical coupling between cells expressing Cx43. Thus, Src utilizes Cas to inhibit gap junctional communication mediated by Cx43. This finding introduces a novel role of the Cas focal adhesion linker protein in the gap junction complex. This observation may help explain how gap junctional communication can be suppressed between malignant and metastatic tumor cells.     

Inhibition of connexin43 gap junctional intercellular communication by TPA requires ERK activation

The phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), is a potent inhibitor of gap junctional intercellular communication (GJIC). This inhibition requires activation of protein kinase C (PKC), but the events downstream of this kinase are not known. Since PKC can activate extracellular signal regulated kinases (ERKs) and these also downregulate GJIC, we hypothesized that the inhibition of GJIC by TPA involved ERKs. TPA treatment (10 ng/ml for 30 min) of WB-F344 rat liver epithelial cells strongly activated p42 and p44 ERK-1 and -2, blocked gap junction-mediated fluorescent dye-coupling, and induced connexin43 hyperphosphorylation and gap junction internalization. These effects were completely prevented by inhibitors of PKC (bis-indolylmaleimide I; 2 microM) and ERK activation (U-0126; 10 microM). These data suggest that ERKs are activated by PKC in response to TPA treatment and are downstream mediators of the gap junction effects of the phorbol ester.     

Gap junctions in human synovial cells and tissue

Our objective was to establish the existence of intercellular communication through gap junctions in synovial lining cells and in primary and passaged cultures of human synovial cells. Communication between cells was assessed using the nystatin perforated-patch method, fluorescent dye transfer, immunochemistry, transmission electron microscopy, and immunoblotting. Functional gap junctions were observed in primary and passaged cultures and were based on measurements of the transient current response to a step voltage. The average resistance between cells in small aggregates was 300 +/- 150 MOmega. Gap junctions were also observed between synovial lining cells in tissue explants; the size of the cell network in synovial tissue was estimated to be greater than 40 cells. Intercellular communication between cultured cells and between synovial lining cells was confirmed by dye injection. Punctate fluorescent regions were seen along intercellular contacts between cultured cells and in synovial membranes in cells and tissue immunostained for connexin43. The presence of the protein was verified in immunoblots. Regular 2-nm intermembrane gap separations characteristic of gap junctions were seen in transmission electron micrographs of synovial biopsies. The results showed that formation of gap-junction channels capable of mediating ionic and molecular communication was a regular feature of synovial cells, both in tissue and in cultured cells. The gap junctions contained connexin43 protein and perhaps other proteins. The physiological purpose of gap junctions in synovial cells is unknown, but it is reasonable to anticipate that intercellular communication serves some presently unrecognized function.     

ZO-1 is required for protein kinase C gamma-driven disassembly of connexin 43

We have previously reported that protein kinase C gamma (PKC-gamma) is activated by phorbol-12-myristate-13-acetate (TPA) and that this causes PKC-gamma translocation to membranes and phosphorylation of the gap junction protein, connexin 43 (Cx43). This phosphorylation, on S368 of Cx43, causes disassembly of Cx43 out of cell junctional plaques resulting in the inhibition of dye transfer. The purpose of this study is to identify the specific role of zonula occludens protein-1 (ZO-1), a tight junction protein with recently established effects on gap junctions, in this PKC-gamma-driven Cx43 disassembly. For this purpose, ZO-1 levels in lens epithelial cells in culture were decreased by up to 70% using specific siRNA. The down-regulation of ZO-1 caused a stable interaction of PKC-gamma with Cx43 even without normal enzyme activation by TPA. However, after TPA activation of the PKC-gamma, the Cx43 did not disassemble out of plaques even though the PKC-gamma enzyme was activated and the Cx43 was phosphorylated on S368. Confocal microscopy demonstrated that the siRNA treatment caused a loss of ZO-1 from borders of large junctional Cx43 cell-to-cell plaques and resulted in the accumulation of Cx43 aggregates inside of cells. Loss of the specific "plaquetosome" arrangement of large Cx43 plaques surrounded by ZO-1 was accompanied by a complete loss of functional dye transfer. These results suggest that ZO-1 is required for Cx43 control, both for dye transfer, and, for the PKC-gamma-driven disassembly response.     

Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.

Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.     

Degradation of Connexins from the Plasma Membrane Is Regulated by Inhibitors of Protein Synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells. Moreover, cell surface connexin saved from destruction by cycloheximide can assemble into long-lived, functional gap junctional plaques. These findings support the concept that downregulation of connexin degradation from the plasma membrane can serve as a mechanism to enhance gap junction-mediated intercellular communication.     

Gap junctional communication is required to maintain mouse cortical neural progenitor cells in a proliferative state

The mechanisms that determine whether neural stem cells remain in a proliferative state or differentiate into neurons or glia are largely unknown. Here we establish a pivotal role for gap junction-mediated intercellular communication in determining the proliferation and survival of mouse neural progenitor cells (NPCs). When cultured in the presence of basic fibroblast growth factor (bFGF), NPCs express the gap junction protein connexin 43 and are dye-coupled. Upon withdrawal of bFGF, levels of connexin 43 and dye coupling decrease, and the cells cease proliferating and differentiate into neurons; the induction of gap junctions by bFGF is mediated by p42/p44 mitogen-activated protein kinases. Inhibition of gap junctions abolishes the ability of bFGF to maintain NPCs in a proliferative state resulting in cell differentiation or cell death, while overexpression of connexin 43 promotes NPC self-renewal in the absence of bFGF. In addition to promoting their proliferation, gap junctions are required for the survival of NPCs. Gap junctional communication is therefore both necessary and sufficient to maintain NPCs in a self-renewing state.     

Phosphorylation of connexin43 on serine368 by protein kinase C regulates gap junctional communication

Phorbol esters (e.g., TPA) activate protein kinase C (PKC), increase connexin43 (Cx43) phosphorylation, and decrease cell-cell communication via gap junctions in many cell types. We asked whether PKC directly phosphorylates and regulates Cx43. Rat epithelial T51B cells metabolically labeled with (32)P(i) yielded two-dimensional phosphotryptic maps of Cx43 with several phosphopeptides that increased in intensity upon TPA treatment. One of these peptides comigrated with the major phosphopeptide observed after PKC phosphorylation of immunoaffinity-purified Cx43. Purification of this comigrating peptide and subsequent sequencing indicated that the phosphorylated serine was residue 368. To pursue the functional importance of phosphorylation at this site, fibroblasts from Cx43(-/-) mice were transfected with either wild-type (Cx43wt) or mutant Cx43 (Cx43-S368A). Intercellular dye transfer studies revealed different responses to TPA and were followed by single channel analyses. TPA stimulation of T51B cells or Cx43wt-transfected fibroblasts caused a large increase in the relative frequency of approximately 50-pS channel events and a concomitant loss of approximately 100-pS channel events. This change to approximately 50-pS events was absent when cells transfected with Cx43-S368A were treated with TPA. These data strongly suggest that PKC directly phosphorylates Cx43 on S368 in vivo, which results in a change in single channel behavior that contributes to a decrease in intercellular communication.     

Roles and regulation of lens epithelial cell connexins

The avascular lens of the eye is covered anteriorly by an epithelium containing nucleated, metabolically active cells. This epithelium contains the first lens cells to encounter noxious external stimuli and cells that can develop compensatory or protective responses. Lens epithelial cells express the gap junction proteins, connexin43 (Cx43) and connexin50 (Cx50). Cx43 and Cx50 form gap junction channels and hemichannels with different properties. Although they may form heteromeric hemichannels, Cx43 and Cx50 probably do not form heterotypic channels in the lens. Cx50 channels make their greatest contribution to intercellular communication during the early postnatal period; subsequently, Cx43 becomes the predominant connexin supporting intercellular communication. Although epithelial Cx43 appears dispensable for lens development, Cx50 is critical for epithelial cell proliferation and differentiation. Cx43 and Cx50 hemichannels and gap junction channels are regulated by multiple different agents. Lens epithelial cell connexins contribute to both normal lens physiology and pathology.     

Acute loss of Cell–Cell Communication Caused by G Protein–coupled Receptors: A Critical Role for c-Src

Gap junctions mediate cell–cell communication in almost all tissues, but little is known about their regulation by physiological stimuli. Using a novel single-electrode technique, together with dye coupling studies, we show that in cells expressing gap junction protein connexin43, cell–cell communication is rapidly disrupted by G protein–coupled receptor agonists, notably lysophosphatidic acid, thrombin, and neuropeptides. In the continuous presence of agonist, junctional communication fully recovers within 1–2 h of receptor stimulation. In contrast, a desensitization-defective G protein–coupled receptor mediates prolonged uncoupling, indicating that recovery of communication is controlled, at least in part, by receptor desensitization. Agonist-induced gap junction closure consistently follows inositol lipid breakdown and membrane depolarization and coincides with Rho-mediated cytoskeletal remodeling. However, we find that gap junction closure is independent of Ca²⁺, protein kinase C, mitogen-activated protein kinase, or membrane potential, and requires neither Rho nor Ras activation. Gap junction closure is prevented by tyrphostins, by dominant-negative c-Src, and in Src-deficient cells. Thus, G protein–coupled receptors use a Src tyrosine kinase pathway to transiently inhibit connexin43-based cell–cell communication.     

Immunoglobulin and cytokine expression in mixed lymphocyte cultures is reduced by disruption of gap junction intercellular communication.

Connexins (Cx), the protein subunits assembled into gap junction intercellular communication channels, are expressed in primary lymphoid organs and by circulating leukocytes. Human tonsil-derived T and B lymphocytes express Cx40 and 43; circulating human T, B, and NK lymphocytes express Cx43 and directly transfer between each other a low molecular dye indicative that functional gap junctions exist. We now identify specific properties in the immune system underwritten by gap junctions. Mixed lymphocytes cultured in the presence of two reagents with independent inhibitory action on gap junction communication, a connexin mimetic peptide and 18-alpha-glycyrrhetinic acid, markedly reduced the secretion of IgM, IgG, and IgA. The secretion of these immunoglobulins by purified B cells was also reduced by the two classes of gap junction inhibitors. Complex temporal inhibitory effects on the expression of mRNA encoding interleukins, especially IL-10, were also observed. The results indicate that intercellular signaling across gap junctions is an important component of the mechanisms underlying metabolic cooperation in the immune system.     

Intercellular calcium signaling via gap junctions in glioma cells

Calcium signaling in C6 glioma cells in culture was examined with digital fluorescence video microscopy. C6 cells express low levels of the gap junction protein connexin43 and have correspondingly weak gap junctional communication as evidenced by dye coupling (Naus, C. C. G., J. F. Bechberger, S. Caveney, and J. X. Wilson. 1991. Neurosci. Lett. 126:33-36). Transfection of C6 cells with the cDNA encoding connexin43 resulted in clones with increased expression of connexin43 mRNA and protein and increased dye coupling, as well as markedly reduced rates of proliferation (Zhu, D., S. Caveney, G. M. Kidder, and C. C. Naus. 1991. Proc. Natl. Acad. Sci. USA. 88:1883-1887; Naus, C. C. G., D. Zhu, S. Todd, and G. M. Kidder. 1992. Cell Mol. Neurobiol. 12:163-175). Mechanical stimulation of a single cell in a culture of non-transfected C6 cells induced a wave of increased intracellular calcium concentration ([Ca2+]i) that showed little or no communication to adjacent cells. By contrast, mechanical stimulation of a single cell in cultures of C6 clones expressing transfected connexin43 cDNA induced a Ca2+ wave that was communicated to multiple surrounding cells, and the extent of communication was proportional to the level of expression of the connexin43 cDNA. These results provide direct evidence that intercellular Ca2+ signaling occurs via gap junctions. Ca2+ signaling through gap junctions may provide a means for the coordinated regulation of cellular function, including cell growth and differentiation.